The frequency of NUDT15 rs116855232 and its impact on mercaptopurine-induced toxicity in Syrian children with acute lymphoblastic leukemia.

Introduction: Polymorphisms in NUDT15 may result in differences in mercaptopurine-induced toxicity. This study aimed to identify the frequency of the NUDT15 (c.415C>T; rs116855232) polymorphism and investigate the effect of this polymorphism on mercaptopurine-induced toxicity in a population of Syrian patients with childhood acute lymphoblastic leukemia (ALL). Methods: This is a retrospective study that included children with ALL reaching at least 6 months of maintenance therapy. The NUDT15 genotyping was determined using standard targeted sequencing of polymerase chain reaction products. The odds ratio (OR) for the association between toxicity and genotype was evaluated. Results: A total of 92 patients were enrolled. The majority of the patients in the study population were low-risk (63.04%), followed by intermediate-risk (25%), and high-risk (11.96%). There were 5 patients (5.4%) with NUDT15 (c.415C>T; rs116855232) CT genotype, and 1 patient (1.08%) with NUDT15 TT genotype, with allele frequencies of C=0.962 and T=0.038. The mercaptopurine median dose intensity was 100%, 54.69%, and 5% for the genotypes CC, CT, and TT, respectively (P=0.009). Early onset leukopenia was significantly associated with the NUDT15 polymorphism (OR: 6.16, 95% CI: 1.11-34.18, P=0.037). There was no association between the NUDT15 genotype and hepatotoxicity. Conclusion: Approximately 6.5% of the study population exhibited reduced NUDT15 activity. The mercaptopurine dose intensity was considerably low in NUDT15 rs116855232 TT genotype compared with CT and CC. The dosage of mercaptopurine should be adjusted according to the NUDT15 genotype in pediatric patients with ALL.

Optimization of tissue microarray technique for breast cancer patients: a short communication.

Background: Tissue microarray (TMA) is a novel technique for studying different types of cancer tissues in one block. TMA is not yet established in Syria, so we aimed in this project to apply and set the most optimal conditions of TMA creation of breast cancer tissues at the Pathology Department of our institute. Materials and Methods: Eighty-eight blocks of breast cancer tissues were selected, considering the inclusion criteria. The tissue specimens of breast cancer patients were manually placed in the block by punching a core from a paraffin block, which was then released into a recipient block using a small trocar. Three different conditions were tested on the constructed TMA block. Results: We determined the most effective parameters that proved high quality: incubating the newly constructed block at a temperature of 43°C for 24 h in the oven and then cutting it the next day after cooling it to room temperature; also, cutting with a 5 µm thickness created the preferable stained slides later. CD3 staining showed high expression of tumor-infiltrating lymphocytes among triple-negative breast cancer patients and high expression of CD3 in triple-negative cancer patients. Conclusion: The optimization of parameters presented in our study resulted in perfect TMA generation and successful immunohistochemistry staining for cancer research at our institution.

The frequencies of CYP2D6 alleles and their impact on clinical outcomes of adjuvant tamoxifen therapy in Syrian breast cancer patients.

BACKGROUND: Tamoxifen is one of the fundamental pillars of adjuvant endocrine therapy for hormone receptor-positive breast cancer; however, 30-50% of patients receiving tamoxifen experience tumor relapse. CYP2D6, encoded by an extremely polymorphic CYP2D6 gene, is the rate-limiting enzyme of tamoxifen bioactivation. This study aimed at determining the frequencies of the most clinically relevant CYP2D6 alleles and evaluating their impact on the responsiveness to tamoxifen in a cohort of Syrian breast cancer patients. METHODS: This case-control study encompassed positive estrogen and/or progesterone receptor, stage 1-3 breast cancer female patients receiving tamoxifen at Al-Bairouni University Hospital, the major National Oncology Center in Syria. Successfully genotyped eligible patients (n = 97) were classified according to their response into; no recurrence group (n = 39) who had completed a five-year recurrence-free adjuvant tamoxifen therapy, and recurrence group (n = 58) who had experienced recurrence. Several star alleles including CYP2D6*4, CYP2D6*10, CYP2D6*41, and CYP2D6*69 were identified via targeted sequencing of specific polymerase chain reaction (PCR) products and phenotypes were assigned according to activity score (AS). The correlation between genotypes and disease-free survival (DFS) was assessed using Kaplan-Meier method and log-rank test. Hazard ratios were estimated using Cox proportional hazards regression models. RESULTS: The allelic frequencies of CYP2D6*41, CYP2D6*10, CYP2D6*4, and CYP2D6*69 were found to be 9.28%, 7.22%, 7.22%, and 2.58%, respectively. No statistically significant differences were observed in the frequencies of CYP2D6 phenotypes between the two arms (P = 0.24), nor the incidence of tamoxifen-induced hot flashes (P = 0.109). Poor metabolizers (PMs) tended to display shorter DFS than intermediate metabolizers (IMs) and normal metabolizers (NMs) combined (adjusted HR = 2.34, 95% CI = 0.84-6.55, P = 0.104). Notably, patients homozygous for the null CYP2D6*4 allele (1847A/A) had an elevated risk of disease recurrence compared to patients with 1847G/G genotype (adjusted HR = 5.23, 95% CI = 1.22-22.49, P = 0.026). CONCLUSIONS: Our findings show no association between CYP2D6 phenotype and treatment outcomes of tamoxifen in Syrian breast cancer patients. Nevertheless, a worse DFS was revealed in patients with 1847A/A genotype (*4/*4).

Platelet lysate induces chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells.

PURPOSE: Articular cartilage has a poor capacity for self-repair, and thus still presents a major challenge in orthopedics. Mesenchymal stem cells (MSCs) are multipotent stem cells with the potential to differentiate into chondrocytes in the presence of transforming growth factor beta (TGF-ß). Platelet lysate (PL) contains a relatively large number of growth factors, including TGF-ß, and has been shown to ameliorate cartilage repair. Here, we investigated the ability of PL to direct chondrogenic differentiation of MSCs along with other standard differentiation components in a pellet culture system. METHODS: We isolated and expanded MSCs from human umbilical cords using a PL-supplemented medium and characterized the cells based on immunophenotype and potential for differentiation to adipocytes and osteocytes. We further cultured MSCs as pellets in a chondrogenic-differentiation medium supplemented with PL. After 21 days, the pellets were processed for histological analysis and stained with alician blue and acridine orange. The expression of SOX9 was investigated using RT-PCR. RESULTS: MSCs maintained their stemness characteristics in the PL-supplemented medium. However, the distribution of cells in the pellets cultured in the PL-supplemented chondrogenic differentiation medium had a greater similarity to cartilage tissue-derived chondrocytes than to the negative control. The intense alician blue staining indicated an increased production of mucopolysaccharides in the differentiated pellets, which also showed elevated expression of SOX9. CONCLUSIONS: Our data suggest that MSCs could be differentiated to chondrocytes in the presence of PL and absence of exogenous TGF-ß. Further research needs to be conducted to understand the exact role and potential of PL in chondrogenic differentiation and chondrocyte regeneration.

Reinforcing the utility of chick embryo model to in vivo evaluate engraftment of human leukemic stem cells.

BACKGROUND AND OBJECTIVE: Development of appropriate translational in vivo models is a prerequisite for personalized management of leukemic patients. Indeed, several immunodeficient mice models were developed for leukemias with main limitations due to their high cost, demanding management, and elongated assessment intervals. In this report, we aimed at evaluating the engraftment of CD34+ cells, isolated from an acute myeloid leukemia (AML) patient, in naturally immunodeficient chick embryo model. METHODS AND RESULTS: Mononuclear cells or immunomagnetic sorted CD34+ cells were injected into chick embryo chorioallantoic membrane (CAM) veins. Seven days post-injection, human CD34 transcript was detected by reverse transcription polymerase chain reaction (RT-PCR) in blood, bone marrow (BM), spleen and liver from embryos injected with human leukemic cells. Interestingly, an amplicon of the same length has been detected in both BM and spleen from PBS injected embryos, although analysis via bioinformatics tools revealed no matches in chicken; neither in transcriptome nor in genome databases. Importantly, splenomegaly and hepatic lesions were observed in some CD34+ cells injected embryos. CONCLUSION: Collectively, our data confirm the engraftment of primary human CD34+ leukemic cells in chick embryo liver, but other experiments are required to verify engraftment in BM and spleen, and to confirm the identity of a putative CD34 orthologous transcript in these two organs.

Identity and synthesis of prostaglandins in the lone star tick, Amblyomma americanum (L.), as assessed by radio-immunoassay and gas chromatography/mass spectrometry.

High concentrations of PGE(2) and PGF(2alpha) were identified by radio-immunoassay (RIA) and/or gas chromatography/mass spectrometry (GC/MS) in the hemolymph, salivary glands and saliva of the lone star tick Amblyomma americanum (L.). Binding studies indicated that PGE(2) was free and not bound to any proteins in the hemolymph. A small amount of 6-keto-PGF(1alpha) (breakdown product of PGI(2); prostacyclin) was also found in the salivary glands but not in the hemolymph or saliva. Neither PGD(2) nor PGA(2)/B(2) was detected in any tick material investigated. Although PGE(2) was found in the gut contents, only small amounts of label crossed the gut into the hemolymph during artificial feeding with labeled PGE(2), indicating that the high amounts of PGE(2) in hemolymph and salivary glands are not sequestered from the host blood meal. Isolated salivary glands and salivary gland homogenates demonstrated robust synthesis of PGE(2) at high concentrations of exogenous arachidonic acid. Synthesis by the salivary glands was monitored by measuring increasing PGE(2) with increasing arachidonic acid by RIA, GC/MS and labeled PGE(2) in the presence of labeled arachidonic acid. Synthesis was inhibited in a dose-dependent manner by indomethacin indicating that the cyclooxygenase synthesizing prostaglandins in ticks shares similarities to the enzyme found in mammals.